JP2023540597A - Conditioning chamber dual interface fluid system - Google Patents

Abstract

The system (100) for investigating membranes (T) comprises a sealed chamber (10) containing a liquid container. The liquid container (11) is configured to hold a liquid medium (M1) in communication with a controlled atmosphere (10a) within the sealed chamber (10). The flow cell (30) is located outside the sealed chamber (10) and comprises flow channels (31, 32) separated by tissue. A set of liquid ducts (L13, L31) is connected to one of the channels (31) and transports the liquid medium (M1) from the liquid container (11) to the barrier (10b) of the sealing chamber (10). , 10t) through the inlet to the channel (30) of the channel (31) to flow at least a portion of the liquid medium (M1') leaving the channel (31) from the channel (30). from the channel (30) back to the liquid container (11). [Selection diagram] Figure 1

Description

The present disclosure relates to a system for interrogating sample membranes and uses of this system.

Microfluidic devices are used to investigate various types of biological and non-biological membranes such as (epithelial) barriers, (fresh) tissue explants, cell layers, scaffolds, or other membranes. can do. For example, a tissue explant can contain different cell types that can be distinguished from a cell monolayer or bilayer, or a suspension of cells injected into a chip. A barrier placed within a microfluidic chip may also be referred to as a "barrier-on-a-chip," eg, "gut-on-a-chip," "gut-on-a-chip," "lung-on-a-chip," "skin-on-a-chip," etc. For example, a "barrier-on-a-chip" allows certain compounds flowing through a first channel (e.g., mimicking the lumen/apical side of a membrane) to pass through a barrier and finally through a second channel. It may be possible to study conditions and extent (e.g., mimicking the blood/basolateral side of the membrane). In some implementations, it is desirable to have anaerobic liquid flow through one channel while having aerobic liquid flow through the other channel to simulate particular conditions in the intestines or lungs, for example. There are cases. In this way, the oxygen content on one side of the epithelial tissue is high and the oxygen content on the other side is low.

By way of background, WO 2017/131839 discloses a microfluidic device aimed at culturing diverse microbial flora, comprising an anaerobic chamber and at least one microfluidic device removably inserted within the anaerobic chamber. Describes the oxygen system. The microfluidic device includes a first microchannel in which a first level of oxygen is maintained and a second microchannel in which a second level of oxygen is maintained; has a higher oxygen concentration than the level of oxygen. The microfluidic device further includes a membrane located in the interfacial region between the first microchannel and the second microchannel, the membrane allowing oxygen to flow from the second microchannel to the first microchannel. The microchannel further includes a plurality of pores forming an oxygen gradient within the microchannel. The system further includes a culture system provided within the first microchannel and containing oxygen sensitive anaerobic bacteria.

There remains a need to further improve known systems, for example to make them more accessible and operable over long periods of time.

One problem with prior art systems is that it can be difficult to monitor the system and take high quality samples over longer periods of time. For example, prior art systems may require a constant flow of anaerobic (nitrogen) gas. It is also necessary to perform anaerobic sampling by placing the entire system in an anaerobic glove box to avoid decomposition of the sample by, for example, oxygen. We take samples and store them under controlled, e.g. anaerobic conditions, even when the system is operated for longer periods without constant resupply of a controlled gas composition. We designed a system that can.

Some aspects of the present disclosure can be embodied as a system for interrogating membranes, eg, tissues. The system includes a conditioning chamber, also referred to herein as a sealing chamber. The chamber includes a barrier, such as a wall and/or a lid that forms an anaerobic box or other airtight container. The barrier is configured to (hermetically) seal the interior of the chamber from the surrounding environment. In this way, a controlled, for example anaerobic, atmosphere within the chamber can be maintained. The liquid container may be placed within the chamber, for example a beaker or other open container placed within the chamber and allowing free exchange of gases. The liquid container is thus configured to hold a liquid medium in open communication with the controlled atmosphere within the chamber. A flow cell, eg a microfluidic chip, is placed outside the first sealed chamber. Flow cells are typically configured to hold tissue across openings between flow channels. In other words, each flow through the channel is separated (exclusively) by the tissue spanning the aperture between them. A first set of liquid ducts, e.g. tubes, is set in one of the flow paths. The duct extends from the first liquid container, through a first barrier, such as a box lid or cover (while maintaining a seal), to the entrance of the first flow path to the flow cell. Configured to carry a liquid medium. Accordingly, a first flow of the first liquid medium through the first flow path along the first side of the tissue can be achieved. At least a portion of the first liquid medium exiting the first flow path from the flow cell is returned to the first liquid container, for example through the same wall or another wall, including inside the first sealed chamber.

By providing the flow cell outside the sealed chamber, the chamber and/or membrane can be easily accessed. By keeping only a portion of the system within a sealed chamber, the chamber can be made relatively small and, for example, the atmosphere within the chamber can be more easily controlled. In addition to the flow cell, other devices for atmospheric measurement and control can also be kept outside the chamber, further improving the accessibility of this device and reducing size requirements. In this way, the chamber can be relatively small compared to systems that need to accommodate all components, such as a large glove box. For example, a sealed chamber can be formed by a closed box that can maintain its atmosphere without constant flushing. By keeping the liquid container open in the sealed chamber, this can allow free exchange of gases forming a first controlled atmosphere with the gases dissolved in the liquid medium. . For example, if the controlled atmosphere is anaerobic, this can cause the liquid medium to become anaerobic as well or remain anaerobic by releasing any oxygen dissolved therein into the controlled atmosphere. It can be done. Furthermore, by recycling the liquid medium into the container, only a limited medium supply is required, which can be constantly exchanged with a controlled atmosphere. Also, by providing sample holders within the sealed chamber, they can be used to receive and hold samples of liquid media under the same controlled atmosphere.

Other or additional aspects of the disclosure may be embodied in methods of using the system. In some embodiments, the use includes providing a liquid medium within a liquid container inside the chamber and providing tissue within a cell connected via a liquid duct to one of the flow channels of the cell. recirculating the liquid medium through the chamber, establishing a controlled atmosphere inside the chamber, and waiting to allow gas in the liquid medium to exchange with the controlled atmosphere; and flowing the liquid medium through the liquid duct through the flow path. Preferably, a controlled atmosphere is established by connecting an atmospheric controller to the valve of the first sealed chamber. Advantageously, after the atmospheric control device has established a controlled atmosphere, the first liquid medium can be disconnected while flowing through the flow cell. Therefore, constant refreshing of the atmosphere, for example by nitrogen flow, is not required.

These and other features, aspects, and advantages of the disclosed devices, systems, and methods will be better understood from the following description, the appended claims, and the accompanying drawings.

FIG. 1 illustrates a system for interrogating membranes. Figure 2A shows a photo of the prototype setup of the system. Figure 2B shows a photo of the prototype setup of the system. FIG. 3A shows a preferred flow cell for tissue placement. Figure B shows a preferred flow cell for tissue placement. Showing various measurements. Showing various measurements. Showing various measurements. Showing various measurements. Showing various measurements. Showing various measurements. Showing various measurements.

The terminology used to describe particular embodiments is not intended to limit the invention. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly dictates otherwise. The term "and/or" includes any and all combinations of one or more of the associated listed items. It is understood that the terms "comprises" and/or "comprising" specify the presence of the stated feature, but do not exclude the presence or addition of one or more other features. Dew. When a particular step of a method is referred to as following another step, unless specified otherwise, said other step can be directly followed or one or more intermediate steps can be performed before performing the particular step. It will be further understood that it can be implemented. Similarly, when a connection between structures or components is described, it will be understood that this connection may be established directly or through an intermediate structure or component, unless otherwise specified.

In some embodiments, the present teachings can be used to provide a gas control chamber that allows creating a dual interface for a perfusion system. This chamber can be used, for example, to reproduce the physiological situation of the intestine, which on the side facing the blood contains a lumen, food and bacteria, with low levels of oxygen; or at a completely anaerobic level. However, the gas mixture in the gas control chamber can be modified to form various interfaces within any (microfluidic) perfusion system. In some setups, the chamber includes an anaerobic medium connected by tubing to a microfluidic chip. The chip contains ex vivo intestinal tissue facing anaerobic media (coming from a gas control chamber) on its luminal side and normal aerobic media (placed in a normal laboratory environment) on its blood facing side. (comes from a drained reservoir). In a peristaltic pump, both anaerobic and aerobic media are recirculated through the tip and reservoir. Decouple the system in the anaerobic glovebox to collect samples by using a flow distributor, selector, or other flow controller (“splitter”) that can split the recirculating medium over different collection tubes. There is no need to open the system or collect anaerobic samples in an aerobic environment in contact with oxygen. The chamber also has a pressure sensor and a pressure regulator, a flexible reservoir such as a balloon or a urine bag, to cope with pressure differences that may arise when placing the gas-controlled chamber in an incubator at 37 degrees from room temperature, for example. can have a connection to. Via another connection, fresh anaerobic air can be injected if necessary. Furthermore, an oxygen sensor can be integrated into or connected to the chamber to monitor the oxygen level within the chamber, but also accurately within the media reservoir. This oxygen sensor can also be placed elsewhere within the microfluidic circuit.

Using recirculation instead of unidirectional perfusion may be more cost effective. There is no need for continuous flushing with nitrogen. Furthermore, since the gas control chamber can be completely closed, any gas mixture can be provided to the chamber using a machine that controls the injection of gas compositions. Multiple possibilities can be provided for connecting the preconditioned fluid in the gas control chamber to the outside world. There is no need to transport the system to an anaerobic glove box to collect samples. For example, the system described herein collects samples in an anaerobic glovebox or in an aerobic environment, and thus anaerobically collects samples in the setup itself, without "contaminating" them with oxygen. It can provide the possibility of sampling. The system can also provide, for example, controlled monitoring of oxygen levels within the chamber and different compartments of the chamber (eg, media reservoirs). The system can also provide a way to sense and regulate pressure differentials inside and outside the gas control chamber.

The invention will be described more fully below with reference to the accompanying drawings, in which embodiments of the invention are shown. In the drawings, the absolute and relative sizes of systems, components, layers, and regions may be exaggerated for clarity. Embodiments can be described with reference to schematic and/or cross-sectional illustrations of sometimes idealized embodiments of the invention and intermediate structures. In this specification and drawings, like numbers refer to like elements throughout. Relative terms and their derivatives should be construed to refer to the orientation as then described or as shown in the drawings under discussion. These relative terms are for convenience of explanation and do not require the system to be constructed or operated in any particular direction, unless explicitly stated otherwise.

FIG. 1 shows a system 100 for investigating a membrane T.

In some embodiments, the system comprises a first sealed chamber 10 with a first barrier 10b, 10t, such as walls and/or a lid, forming an anaerobic box or other airtight container. Preferably, the first sealed chamber 10 is configured to (hermetically) seal the interior of the first sealed chamber 10 from the surrounding environment E. In this way, a first controlled atmosphere 10a can be maintained inside the first sealed chamber 10.

In some embodiments, a first liquid container 11 is housed inside the first sealed chamber 10, for example a beaker or other open container placed within the chamber and allowing free exchange of gases. It is. For example, the first liquid container 11 holds a first liquid medium M1 in the first sealed chamber 10 that communicates with the first controlled atmosphere 10a.

In some embodiments, a flow cell 30, eg, a microfluidic cell, is placed outside of the first sealed chamber 10. Preferably, the flow cell 30 includes at least a first flow path 31 and a second flow path 32. In one embodiment, a clamping structure 33 or other interface is configured to hold the membrane T spanning the opening between the channels 31, 32. The respective flows through channels 31, 32 are thus separated by a membrane T spanning the opening between them.

In some embodiments, a first set of liquid ducts L13, L31, e.g. tubes, are connected to the first flow path 31. For example, the liquid duct transports the first liquid medium M1 from the first liquid container 11 through the first barrier 10b, 10t (while maintaining a seal), for example the lid or cover of a box, into the first flow path. 31 to the entrance to the flow cell 30. In this way, the duct can facilitate a first flow of the first liquid medium M1 along the first side of the membrane T through the first flow path 31. In a preferred embodiment, a liquid duct carries at least a portion of the first liquid medium M1' leaving the first flow path 31 from the flow cell 30, for example on the same wall 10t comprising the inside of the first sealed chamber 10 or elsewhere. through the wall of the first liquid container 11 .

In some embodiments, the system comprises a second liquid container 21 configured to hold a second liquid medium M2. Typically, the second liquid medium M2 has a different composition than the first liquid medium M1. For example, the second liquid medium M2 has different (amounts) of solutes, eg oxygen, or other components such as nutrients, medicines, etc. Optionally, the medium itself may be formed, for example, by different solvents.

In some embodiments, a second set of liquid ducts L23, L32 is connected to the second flow path 32 and transports the second liquid medium M2 from the second liquid container 12 to the inlet of the second flow path 33. and configured to convey a second flow of a second liquid medium M2 along a second side of the membrane T through a second flow path 32. Preferably, the second set of liquid ducts L23, L32 is further configured to return at least a portion of the second liquid medium M2' exiting the second flow path 32 from the flow cell 30 to the second liquid container 21. Ru. In other words, at least a portion of the second liquid medium can also be recycled. Alternatively, some or all of the first and/or second liquid medium can be discarded after traversing the flow cell 30.

For example, as shown, the first flow path through the first set of liquid ducts L13, L31 has a first flow path length through the first flow path 31 and back to the first flow path 11. However, a second flow path length that passes through the second set of liquid ducts L23 and L32, passes through the second flow path 32, and returns to the second liquid container 21 has a second flow path length. In a preferred embodiment, the first and second path lengths are essentially the same, for example within 10 percent, preferably within 5 percent, or less, i.e. substantially the same (within measurement tolerances). ). By providing similar path lengths (and similar or same fluid ducts), flow can be similar on both sides of the tissue. This may, for example, prevent pressure or other differences between the flows that could affect tissue measurements.

In some embodiments, the system comprises or is coupled to a pump 40 disposed in the path of the first and/or second set of liquid ducts. For example, pump 40 is configured to control the respective flow through respective channels 31, 32 of flow cell 30. Typical flows may be set, for example, from 1 to 100 milliliters per hour. Other flow rates may also be used depending on, for example, the flow cell 30 and the sample under study. In some embodiments, the same pump is used to control the respective flows within both sets of liquid ducts. Also, a separate pump can be used. For example, one or more pumps can be used to set the same or different flow rates in each channel 31, 32. In some embodiments, for example, as shown, the pump 40 is positioned along the respective liquid duct L31, L32 in the flow path after the respective outlet of the flow cell 30 back to the respective liquid container 11, 12. Ru. For example, a pump is placed between the flow cell 30 and the flow distributors 15, 25 (described in more detail below). Alternatively, the pumps can be placed elsewhere, for example in the respective liquid ducts L13, L23 in the flow path before the flow cell. Preferably the pump is a peristaltic pump, also known as a roller pump. For example, the fluids are contained within flexible tubes that form at least part of the respective liquid ducts. The flexible tube can be attached inside the pump casing. In circular pump casings, a rotor with multiple "rollers," "shoes," "wipers," or "lobes" attached to the outer circumference of the rotor can be used to compress the flexible tube. As the rotor rotates, the portion of the tube that is under compression is pinched shut, thereby forcing the pumped fluid to move through the tube. In addition, the tube opens when the tube opens to its natural state after a flow of cam fluid is induced into the pump. There are typically two or more rollers or wipers that occlude the tube and trap the body of fluid between them. Linear peristaltic pumps can also be used.

In some embodiments, the system includes a second sealed chamber 20. For example, the second sealed chamber 20 includes a second barrier configured to seal the interior of the second sealed chamber 20 from the surrounding environment E. Therefore, a second controlled atmosphere 20a can be maintained inside the second sealed chamber 20. In other or further embodiments, the second liquid container 21 is housed inside the second sealed chamber 20. For example, the second liquid container 21 holds a second liquid medium M2 within the second sealed chamber 20 that communicates with the second controlled atmosphere 20a. In other words, the system may include multiple seal chambers that may be similar or identical. For example, each channel 31, 32 of the flow cell 30 is connected to a respective sealed chamber 10 with a respective liquid container 11, 21 containing a respective liquid medium M1, M2 exchanging with a respective controlled atmosphere 10a, 20a. , 20. Typically, the atmosphere within each chamber is controlled to have a different atmosphere. For example, the first controlled atmosphere may have an anaerobic, relatively low oxygen content, while the second controlled atmosphere may have an aerobic, relatively high or normal oxygen content.

Instead of sealing the second liquid container 21 in the respective sealing chamber 20, the second liquid container 21 or at least its sealing can be omitted. For example, the second liquid container 21 can remain in open communication with the surrounding environment E. For example, if the surrounding environment E is aerobic, the second liquid medium M2 can be made aerobic as well by exchanging the oxygen of the environment with the second liquid medium M2. A closed enclosure holding the second liquid medium M2 with or without recirculation flow may also be envisaged. Alternatively or additionally, it may also be envisaged that the second liquid container 21 is accommodated in the same first sealed chamber 10 as the first liquid container 11. For example, both the first liquid medium M1 and the second liquid medium M2 can be exposed to the same first controlled atmosphere 10a. For example, both liquids M1, M2 are kept under the same atmospheric conditions, but their eg non-gas content may differ in some other way that may be independent of these conditions.

In some embodiments, the system includes a first flow distributor 15 or divider connected to a first set of liquid ducts L13, L31 between the first flow path 31 and the first sealed chamber 10. Equipped with a splitter/splitter. Preferably, the first flow distributor 15 transfers the incoming flow of the first liquid medium M1' at the inlet port 15a of the first flow distributor 15 to the inlet flow of the first liquid medium M1' at the respective outlet port 15b of the first flow distributor 15. and configured to selectively and/or controllably direct one or more outlet streams. In a preferred embodiment, at least one of the outlet ports 15b is routed through the first barrier 10b, 10t to direct at least a portion of the first liquid medium M1' back into the first liquid container 11. Connected to liquid duct. For example, each outlet port is provided with one or more controllable valves 15v to open and close the respective outlet flow. For example, the valve can be formed by a releasable clamp on a tube forming a duct into the container. Also, other types of valves may be envisaged.

In some embodiments, the first sealed chamber 10 includes one or more chambers configured to receive and hold a liquid sample of the first liquid medium M1 (separate from the first liquid container 11). sample container 12. For example, at least one of the outlet ports 15b of the first flow distributor 15 (selectively) directs at least a portion of the first liquid medium M1' into a respective sample container of the one or more sample containers 12. For directing, it is connected to a liquid duct passing through the first barrier 10b, 10t. For example, a respective valve 15v at the outlet 15b of the first flow distributor 15 is operative to control flow into the first liquid container 11 and/or into one or more of the sample containers 12. can be done. Advantageously, the sample container 12 is provided with a first controlled atmosphere 10a capable of preventing deterioration of the respective sample taken from the flow of the first liquid medium M1 passed through the first channel 31, for example. can be held within. By providing a plurality of sample containers 12a-12d inside the first sealed chamber 10, different samples of the first liquid medium M1 can be taken, for example at different times. Similarly, the second liquid medium M2 can also be sampled. For example, a second flow distributor 25 is connected to the second set of liquid ducts L23, L32 and directs the flow of the second liquid medium M2 into the second liquid container 21 and/or into one or more sample containers. 22. The sample container 22 may be sealed together with the second liquid container 21 within the second sealing chamber 20 or simply kept in the ambient environment E, depending on the desired conditions.

Naturally, the ducting through the barrier of each sealing chamber is configured to prevent exchange of gases within the controlled atmosphere (contained within the chamber by the barrier) with the surrounding environment E. In other words, each duct forms a gas-tight connection with a respective barrier of the sealing chamber. For example, the liquid duct is configured to exclusively allow the liquid medium inside the duct to pass through the barrier while preventing leakage of the surrounding environment E. Also, other sealed ducts can be used, as described below.

In some embodiments, the system includes a pressure control system 16 configured to maintain a controlled pressure of the first controlled atmosphere 10a within the first sealed chamber 10. Preferably, the pressure control system 16 is configured to maintain the same pressure inside the first sealing chamber 10 as the surrounding environment E. For example, the first controlled atmosphere 10a inside the first sealing chamber 10 may be at the same or similar pressure (e.g. measured in Bars or Pascals) as the surrounding environment E of the first sealing chamber 10, e.g. It is maintained within ±5%, preferably within ±2%, more preferably within ±1% or less of the ambient air pressure. This can relieve the external atmosphere leaking into the chamber, or vice versa, and can also relieve, for example, non-uniform pressures on the sample membrane. The pressure control system can be relatively simple, e.g. with a flexible barrier surface (preferably with minimal elasticity) capable of relaying pressure from the environment to the interior of the container without exchanging gases. be able to. For example, the flexible bag can be connected to the controlled atmosphere 10a inside the sealed chamber 10 via a duct. Alternatively, the walls of the first sealed chamber can be formed by flexible sheets that can move freely to compensate for any pressure differences but are impermeable to gas to maintain a controlled atmosphere 10a. It can also be assumed that

In some embodiments, the system includes an atmospheric control valve 13 passing through the first barrier 10b, 10t, and an atmospheric controller (not shown) to establish a first controlled atmosphere 10a inside the first seal 10. (removably) to the first seal 10. For example, the atmospheric control valve 13 includes a snap-shut connection or a threaded connection for removably connecting an atmospheric control device.

In one embodiment, when the atmospheric control device is connected to the atmospheric control valve 13, the atmospheric control valve 13 connects the exchange duct through the first barrier 10b, 10t to initially establish the first controlled atmosphere 10a by the atmospheric control device. configured to open. In another or further embodiment, upon disconnection of the atmospheric control device from the atmospheric control valve 13, the atmospheric control valve 13 is configured to close the exchange duct for maintaining the first controlled atmosphere 10a in the absence of the atmospheric control device. Ru.

In some embodiments, the system includes an atmospheric controller configured to establish and/or maintain a first controlled atmosphere 10a within the first sealed chamber 10. An example of such an atmospheric controller is commercially available as the Anoxomat® III, which can be purchased from Advanced Instruments. For example, a controller can be used to supply a gas mixture and form a sealed chamber. For example, an "A-box" (available from Kentron Microbiology) can be used with a snapshot coupling to connect an atmospheric controller. We adapted the box to serve the current purpose of a sealed chamber by designing an adapted lid with airtight holes to fit conduits such as liquid/gas ducts, sensor fibers, etc. I let it happen. For example, the chamber may be adapted to have multiple fittings that accommodate high pressure connections (which are standard) through the wall (lid) of the chamber. In some embodiments, the second sealed chamber 20 may be connected to a similar or other atmosphere controller to establish the same or a different controlled atmosphere 20a. Alternatively, the second sealed chamber 20 can remain in open communication with the surrounding environment E or can be omitted entirely.

In principle, the first sealed chamber 10 can be sealed sufficiently to maintain the atmosphere after disconnecting the controller. If necessary, the atmosphere can be replenished by the same controller or by other means. In some embodiments, the system comprises an atmosphere (re)supply unit 17 configured to supply and/or replenish the first controlled atmosphere 10a within the first sealed chamber 10. For example, the atmospheric supply unit 17 can include a supply of controlled atmospheric air 10a that can be supplied to the first sealed chamber 10. The atmospheric supply unit 17 can advantageously be combined with a pressure control system 16, so that the supply of excess gas preferably does not change the pressure in the first sealed chamber 10.

In some embodiments, the system measures, e.g., pressure and/or oxygen, reports the readout, and optionally includes one for feedback and control coupled to, e.g., one or more reservoirs. configured to connect with one or more sensors. For example, the system comprises a sensor 18 configured to measure the first liquid medium M1 in the first liquid container 11, for example. For example, the sensor is configured to measure the concentration of oxygen and/or other substances in the first liquid medium M1. Advantageously, this can be combined with an atmospheric supply unit 17, for example allowing extra gas to be supplied into the first sealed chamber 10 in response to measurements. For example, if the oxygen content of the first liquid medium M1 is determined to be too high, the atmospheric supply unit 17 can be used to supply nitrogen (without oxygen) into the sealed chamber 10. Also, other or additional sensors can be provided in a similar manner. For example, a pressure sensor can include a pressure sensing element held within a first controlled atmosphere 10a. Advantageously, the majority of the sensors can be placed outside the sealing chamber 10, e.g. providing only a measuring fiber or other wire through the barrier 10b, 10t (while maintaining a controlled atmosphere 10a seal). can do.

Also, other or additional means may be provided to maintain a controlled atmosphere inside the sealed chamber. For example, oxygen binding or absorbing materials can be provided within the chamber to establish and/or maintain an anaerobic atmosphere. As will be appreciated, such measures may be particularly effective if the volume of the chamber is relatively small.

In preferred embodiments, the first sealed chamber 10 has a volume (maximum internal volume) of less than 100 liters, less than 50 liters, less than 20 liters, less than 10 liters, or even less than 5 liters. The smaller the chamber internal volume, the easier it may be to establish and/or maintain a controlled atmosphere. It is noted that part or most of the internal volume within the chamber may be taken up by a liquid container together with a liquid medium and/or a sample holder. Therefore, the actual amount of air to be controlled may be even less. For example, the chamber has a maximum internal dimension of less than 50 centimeters, less than 30 centimeters, or even less than 20 centimeters.

In some embodiments, the system includes a first flow distributor connected to each outlet port 15b of the first flow distributor 15 and configured to selectively receive at least a portion of the first liquid medium M1'. A waste container W1 is provided. Preferably, the first waste container W1 is arranged outside the first sealed chamber 10. For example, a waste container can be used to capture an initial or other stream of first liquid medium M1 that does not need to be recycled and can be disposed of. For example, the initial flow may contain oxygen or contaminants that may initially be present within the first flow path 31 and/or the first set of liquid ducts L13, L31. Therefore, the first waste container W1 is preferably not kept inside the first sealed chamber 10 and may contaminate the first controlled atmosphere 10a and/or take up unnecessary space. be. Similarly, the second flow distributor 25 can also be connected to a second waste container W2, which can be kept in the ambient environment E. Optionally, the same waste container can be used for both streams.

In some embodiments, the system 100 described herein can be used as follows. A first liquid container 11 in the first sealed chamber 10 is provided with a first liquid medium M1. The stream 30 connected via the first set of liquid ducts L13, L31 is provided with a membrane T for recirculating the first liquid medium M1 via the first channel 31 of the stream. There is. The first controlled atmosphere 10a in the first sealed chamber 10 is established and there may be a waiting period for allowing the gas in the first liquid medium M1 to be exchanged with the first controlled atmosphere 10a. After this, the first liquid medium M1 can flow through the first channel 31 of the flow cell 30 via the first set of liquid ducts L13, L31. Preferably, the first controlled atmosphere 10a is established by connecting an atmospheric control device to the atmospheric control valve 13 of the first sealed chamber 10. For example, after the atmospheric control device establishes the first controlled atmosphere 10a, it is disconnected while allowing the first liquid medium M1 to flow through the flow cell 30. In some embodiments, the first controlled atmosphere 10a is anaerobic, such as less than 1 percent, preferably less than half of 1 percent, or even less than one-tenth of 1 percent, such as 0.05%. It has an oxygen concentration of In one embodiment, the concentration of oxygen in the first liquid medium M1 is measured while flowing the first liquid medium M1 through the flow cell 30. For example, upon determining that the concentration of oxygen is higher than a predetermined threshold, a further supply of anaerobic air is injected into the first sealed chamber 10. Other uses of the device can also be envisaged.

The systems described herein can be used, for example, to measure, test, and/or analyze the permeability of substances through membranes. For example, membranes include (epithelial) barriers, (fresh) tissue explants, cell layers, scaffolds, or other membranes. For example, a tissue explant can contain different cell types that can be distinguished from a cell monolayer or bilayer, or a suspension of cells injected into a chip. In use, the membrane is placed in the cavity opening between channels 31, 32. For example, the substance can be disposed in at least one of the fluid streams, eg, in different amounts. In some embodiments, the content of each of the substances in the first and/or second reservoir streams is measured and/or monitored to determine the exchange of substances through the membrane. For example, substances may include nutritional ingredients, pharmaceuticals, and the like. For example, membranes include tissue samples, scaffolds, eg, 3D structures with cells, or other membranes, eg, plastic or polyester.

2A and 2B show photographs of a prototype setup of the system described herein. FIG. 2A shows a first chamber 10 that is closed and a second chamber 20 that is open. In this case, two flow cells 30 containing ex vivo intestinal tissue are connected via respective tubes and peristaltic pumps 40. Figure 2B shows further details of the first chamber 10, including the lid 10t, which is hermetically connected to the box below it. Besides the gas valve 13, the lid has several sealing openings to allow ducts and fibers to pass therethrough. For example, a flow distributor 15 as shown is connected to multiple sample containers within chamber 10. Each flow to one of the sample holders can be selected by opening a respective one of the fluid valves 15v.

FIG. 3A shows a perspective view of a flow cell 30 (microfluidic device) closed by a cap 35. In one embodiment, for example as shown, the flow cell 30 has a housing defining at least two flow passages 31, 32. Also, more than two channels may be envisaged (not shown). In the illustrated embodiment, the flow cell 30 is translucent so that the channels 31, 32 through the housing 38 as well as the possible contents of the channels are visible. This allows easy viewing of the flow inside the housing without opening the cap. Also, other, eg opaque materials can be used. Preferably, housing 38 consists essentially of a material such as, for example, plastic. In a preferred embodiment, for example, as shown herein, the device is manufactured by 3D printing. For example, the device is made from a printable (cured) material. Also, other manufacturing methods and materials can be envisaged.

In one aspect, the present disclosure provides a flow cell 30 for analyzing the permeability of a substance through a (sample) membrane T, such as tissue. For example, substances are placed in respective fluid streams. Typically, flow cell 30 includes a housing 38 having flow channels 31,32. In some embodiments, first flow path 31 is configured to pass a first fluid flow through housing 38 between first input connector 31a and first output connector 31b. In other or further embodiments, the second flow path 12 is configured to communicate a second fluid flow through the housing 38 between the second input connector 32a and the second output connector 32b.

In a preferred embodiment, the device comprises an access cavity extending through the first flow path 11 into the second flow path 12 and into the housing 38 from the outside. The cavity can thus be used to access the inside of the housing 38 in order to place the membrane T over the cavity opening 33. Most preferably, the cavity opening 33 forms an (exclusive) fluid interconnection between the overlapping regions of the channels 31, 32. In a preferred embodiment, the access cavity can be opened and closed by a cap 35. Most preferably, the cap 35 can be reversibly removed to access the cavity and, for example, to place or replace the membrane T. For example, the cap 35 and/or the housing include a resilient material such as rubber between the seal and the housing. Also, other elastic structures can be used or the cap can be screwed onto the housing.

FIG. 3B shows a cutaway/exploded view of flow cell 30 with various components. In a preferred embodiment, flow cell 30 includes a clamp ring 34. In one embodiment, for example, as shown, clamp ring 34 includes a connecting structure. For example, the connection structure is configured to engage a corresponding connection structure of the housing within the access cavity. The clamp ring 34 can therefore be used to hold the sample membrane T in place above the cavity opening 33. Typically, when the membrane T is in use, it is exposed to respective fluids flowing through channels 31, 32 on either side of the membrane T. In the preferred embodiment the opening is circular, but other shapes can be envisaged. Typically, sealing ring 36 is configured to contact membrane T (in use).

In one embodiment, the housing 38 extends with the cavity seat to form a platform around the cavity opening 33 between the overlapping regions of the channels 31, 32, and a membrane T between the cavity seat and the clamp ring 34. directly or indirectly. For example, the membrane T can be placed directly on the cavity seat. For example, the clamp ring 34 can clamp the membrane T directly. Preferably a sealing ring 36 is arranged on one or both sides of the membrane T. In one embodiment, at least one seal ring 36 is disposed between the membrane T and the clamp ring 34 in use, for example as shown. This may improve the seal and/or prevent the clamp ring from damaging (eg, cutting) the membrane T. Also, other or additional structures may be placed between the clamp ring 34 and the membrane T and/or between the membrane T and the cavity seat. For example, a mesh can be provided to help further support the membrane T. In some embodiments, for example, as shown, the membrane T is disposed between the seal ring 36 and the cavity seat. Other or further configurations may also be envisaged. In one embodiment, a ring opening through the cavity opening 33 on one side of the membrane T and the clamp ring 34 on the other side of the membrane T allows the membrane T to flow in the respective fluid flow paths 31, 32 in use. configured to expose the

4A-4C show the oxygen concentration (O ₂ %) as a function of time (T in time [h]). FIG. 4A shows the oxygen concentration in the air forming the atmosphere 10a of the sealed chamber, and FIG. 4B shows the oxygen concentration in the liquid medium M1 accommodated in the sealed chamber. After the "Anoxomat" procedure to make the gas control chamber anaerobic, the air (e.g. with the help of a catalyst in the chamber that scavenges oxygen) drops even more rapidly to less than 0.20%. It has oxygen concentration. After the procedure, medium M1 is still rich in oxygen, which rapidly drops to a level below 0.20% over the next 12 hours. The oxygen-rich air in the medium diffuses with the anaerobic air in the chamber.

Figure 4C shows the oxygen percentage measured in the anaerobic medium inside the anaerobic device during control experiments with tubes and empty tips. After 24 hours of making the gas-controlled chamber and the medium inside the chamber anaerobic, it was connected to tubing, a microfluidic chip (without intestinal tissue), and a peristaltic pump. The system was run at 2 ml/hr for 72 hours. Except for the initial slight increase in oxygen level due to suction of air from the splitter (probably a result of the under pressure in the box and not enough connections of the plumbing in the splitter being made, this (later improved), the anaerobic medium in the chamber remained anaerobic for 72 hours.

After these so-called "dry" experiments (which do not involve intestinal tissue or other biological materials connected to the system), experiments with intestinal tissue attached to microfluidic chips have also been conducted. From these initial experiments it was observed that the system could be sensitive to pressure differences inside and outside the chamber. For example, pressure differences between the gas control chamber and atmospheric pressure can cause problems in microfluidic chips (leakage of large fluorescent molecules through the tissue and cross-flow from the apical to the basolateral medium, and its Tissue damage reflected by the inverse). Therefore, it is preferred to use means for sensing and/or regulating the air pressure.

Figures 5A and 5B show the pressure difference observed inside the system compared to atmospheric pressure in two different experiments. FIG. 5A shows the difference in cm measured using a hydrostatic physical test. Figure 5B shows the difference measured using an external reservoir. The chamber had atmospheric pressure after the Anoxomat procedure, but when placed in the 37 degree incubator the pressure increased, but later dropped again, for example as the catalyst consumed the last bit of oxygen. Flush the system of media or keep it at room temperature while at low pressure. The pressure difference can be adjusted by a flexible air reservoir and by injecting air, for example via a syringe. It was observed that recirculating and sampling the starting medium within the chamber by using different splitter outlets did not cause any additional pressure differences (points 5, 6, 7 in Figure 5B).

One purpose of this prototype system is to be able to expose the intestinal membrane to strictly anaerobic bacteria living in the intestine and study host-microbe interactions. Since these bacteria survive only under strictly anaerobic environments (some are already killed at oxygen levels >0.5%), anaerobic bacterial strains can survive in our system. An experiment was performed to assess whether (intestinal tissue, no medium with bacteria passing through the tube at 2 ml/h). Two different media were tested, Williams E and SIEM. Bacteria cultured in SIEM medium survived and showed growth during 24 hours, whereas bacteria cultured in Williams E died (Table 1). As shown in Figures 6A and 6B, oxygen levels remained low in both Williams E and SIEM.

It is to be understood that, in interpreting the appended claims, the word "comprising" does not exclude the presence of other elements or acts other than those recited in a given claim; The word "a" or "an" does not exclude the presence of more than one such element; any reference signs in the claims do not limit their scope; several "means" do not exclude the same or different items; Any of the disclosed devices or portions thereof may be combined together or further portions unless stated otherwise. When one claim refers to another claim, this may indicate a synergistic advantage achieved by the combination of their respective features. However, the mere fact that certain measures are recited in mutually different claims does not indicate that a combination of these measures cannot also be used to advantage. Accordingly, the present embodiments may include all operating combinations of the claims, which in principle can refer to any preceding claim, unless each claim is clearly excluded by the context.

Claims (15)

A system (100) for investigating a membrane (T), comprising:
a first sealed chamber (10), the first sealed chamber (10) configured to maintain a first controlled atmosphere (10a) within said first sealed chamber (10); a first sealed chamber (10) having a first barrier (10b, 10t) configured to seal the interior of the chamber (10) from the surrounding environment (E);
A first liquid container (11) housed inside the first sealed chamber (10), wherein the first liquid container (11) is housed inside the first sealed chamber (10). a first liquid container (11) configured to hold a first liquid medium (M1) in open communication with said first controlled atmosphere (10a) at
A flow cell (30) disposed outside the first sealed chamber (10), the flow cell (30) having a first flow path (31) and a second flow path (32). , a clamp structure (33) configured to hold the membrane (T) stretched over the opening between the channels (31, 32), and the membrane (T) a flow cell (30) configured to separate each flow through the flow path (31, 32) with an opening in between;
connected to the first flow path (31), conveying the first liquid medium (M1) from the first liquid container (11) and passing through the first barrier (10b, 10t) to the first liquid medium (M1). 1 flow path (31) to the inlet of the flow cell (30), and the first liquid medium (M1') exiting from the first flow path (31). ) from the flow cell (30) back to the first liquid container (11);
a first flow distributor (15) connected to the first set of liquid ducts (L13, L31) between the first flow path (31) and the first sealed chamber (10); selectively converting an inlet stream of said first liquid medium (M1') into a respective outlet stream at a respective outlet port (15b) of said first flow distributor (15). a first flow distributor (15) configured to direct;
Equipped with
At least one of said outlet ports (15b) comprises said first barrier ( 10b, 10t) connected to the liquid duct,
Said first sealed chamber (10) is further arranged in said first controlled atmosphere (10a) inside said first sealed chamber (10), said first liquid container (11) one or more sample containers (12) configured to receive and hold a liquid sample of said first liquid medium (M1), separate from said first flow distributor (15); ) for directing at least a portion of the first liquid medium (M1') into one or a respective sample container of the plurality of sample containers (12). one or more sample containers (12) connected to a liquid duct passing through a first barrier (10b, 10t);
system. System according to claim 1, wherein the first sealed chamber (10) has a volume of less than 5 liters. a second liquid container (21) configured to hold a second liquid medium (M2);
The second liquid medium (M2 ) a second set of liquid ducts (L23, L32) configured to carry
Equipped with
Said second set of liquid ducts (L23, L32) directs at least a portion of said second liquid medium (M2') exiting said second flow path (32) from said flow cell (30) to said second liquid medium (M2'). further configured to return the liquid container (21) to the liquid container (21) of the
The system according to claim 1 or claim 2. a second sealed chamber (20), the second sealed chamber (20) comprising: a second sealed chamber (20) including a second barrier configured to seal the interior of the sealed chamber (20);
a second liquid container (21) is housed inside the second sealed chamber (20);
The second liquid container (21) is configured to hold a second liquid medium (M2) within the second sealed chamber (20) in open communication with a second controlled atmosphere (20a). has been,
System according to any one of claims 1 to 3. A first passage through the first set of liquid ducts (L13, L31) from the first liquid container (11) through the first flow path (31) and back to the first liquid container (11). the flow path has a first path length;
The second liquid duct passes through a second set of liquid ducts (L23, L32) from the second liquid container (21) through the second flow path (32) and returns to the second liquid container (21). the flow path has a second path length;
the first and second path lengths are essentially the same;
The system according to claim 4. disposed in the path of the first set of liquid ducts and/or the second set of liquid ducts to control the respective flow through each of the flow channels (31, 32) of the flow cell (30); a peristaltic pump (40) configured;
System according to any one of claims 1 to 5. a pressure control configured to maintain the controlled pressure of the first controlled atmosphere (10a) inside the first sealed chamber (10) the same as the pressure of the surrounding environment (E); comprising a system (16);
System according to any one of claims 1 to 6. an atmospheric control device for establishing and/or maintaining a first controlled atmosphere (10a) through the first barrier (10b, 10t) and inside the first sealed chamber (10); configured to connect to said first sealed chamber (10);
comprising an atmospheric control valve (13);
System according to any one of claims 1 to 7. Any one of claims 1 to 8, comprising an atmospheric controller configured to establish and/or maintain said first controlled atmosphere (10a) inside said first sealed chamber (10). The system described in Section. When an atmosphere control device is connected to an atmosphere control valve (13), said atmosphere control valve (13) controls said first controlled atmosphere (10a) for initially establishing said first controlled atmosphere (10a) by said atmosphere control device. configured to open the exchange duct through the barrier (10b, 10t).
A system according to claim 8 or claim 9. When the atmospheric control device is disconnected from the atmospheric control valve (13), the atmospheric control valve (13) is connected to the exchange for maintaining the first controlled atmosphere (10a) in the absence of the atmospheric control device. Configured to close the duct.
System according to claim 10. a sensor (18) configured to measure the concentration of oxygen in the first liquid medium (M1) in the first liquid container (11);
comprising an atmospheric resupply unit (17) configured to supply and/or replenish anaerobic air inside said first sealed chamber (10) on the basis that the measured concentration exceeds a threshold value; ,
System according to any one of claims 1 to 11. a first waste connected to a respective outlet port (15b) of said first flow distributor (15) and configured to selectively receive at least a portion of said first liquid medium (M1'); A storage container (W1) is provided.
the first waste container (W1) is arranged outside the first sealed chamber (10);
System according to any one of claims 1 to 12. providing a first liquid medium (M1) in a first liquid container (11) inside a first sealed chamber (10);
providing a membrane (T) within a flow cell (30) connected via a first set of liquid ducts (L13, L31); recirculating the liquid medium (M1);
establishing a first controlled atmosphere (10a) inside said first sealed chamber (10);
waiting for gas in said first liquid medium (M1) to exchange with said first controlled atmosphere (10a);
causing said first liquid medium (M1) to flow through said first channel (31) of said flow cell (30) via said first set of liquid ducts (L13, L31); ,
Use of a system according to any one of claims 1 to 13, comprising: said first controlled atmosphere (10a) is established by connecting an atmospheric control device to an atmospheric control valve (13) of said first sealed chamber (10);
After the atmospheric control device establishes the first controlled atmosphere (10a), the first sealed chamber (10) is flown while the first liquid medium (M1) is flowing through the flow cell (30). be separated from
Use according to claim 14.